Pullulanase is an enzyme that hydrolyzes .alpha.-1, 6-glucosidic bond of pullulan to eventually produce maltotriose. Recently, pullulanase has drawn a special attention due to its enzymatic ability to remarkably increase the production of various saccharides: pullulanase is used along with glucoamylase (optimum pH: 4.5, optimum temperature: 60.degree. C.) to improve the yield of glucose from starch; pullulanase is used along with soybean .beta.-amylase [optimum pH: about 6, optimum temperature: 55.degree.-60.degree. C., Enzyme and Food Processing ed. by G. G. Brich, Applied Science Publishers Ltd., London, 73-88 (1981) Japanese Patent Application Kokoku 37849/1971 etc.] or bacterial .beta.-amylase [optimum pH: about 7, optimum temperature; about 50.degree. C., Y. Takasaki, Agric. Biol, Chem., Vol. 40, No. 8, 1523 (1976) etc.] to obtain higher yield of maltose from starch; pullulanase is used along with enzymes (each of them has optimum pH: about 7-8, optimum temperature: 55.degree.-60.degree. C.) which are produced by bacteria belonging to genus Bacillus etc. to improve the yield of maltooligosaccharides such as maltotriose, [Y. Takasaki, Agric. Biol. Chem., Vol. 49, No. 4, 1091 (1985) etc.] maltotetraose [J. F. Robyt et. al., Arch. Biochem., Biophys., Vol. 145, 105 (1971), Y. Takasaki, Agric. Biol. Chem., Vol. 55, No. 7, 1715 (1991) etc.] maltopentaose [N. Saito, Arch. Biochem. Biophys., Vol. 155, No. 290 (1978) etc.] and maltohexaose [K. Kainuma et. al., J. Biochem. Biophy. Acta, Vol. 410, 333 (1975), Y. Takasaki, Agric. Biol. Chem., 46, 1589 (1982) Fermentation and Industry, Vol. 40, 477 (1983) etc.].
The working pH of these amylase used along with pullulanase is in the range of as large as 4-8 so that pullulanase is required to be enzymatically active in the same pH range for the production of saccharides. In addition, the enzyme should be heat stable, for example, at least 55.degree.-60.degree. C., during reaction to prevent the contamination of microorganisms, and should be used for a long period of time at that temperature for the industrial application of the enzyme.
It has been known that pullulanase is produced by many kinds of bacteria such as those belonging to genus Aerobacter [Aerobacter aerogenes (Klebsiella pneumoniae), H. Bender and K. Wallenfels, Biochem. Z. Vol. 334, 79(1961), M. Abdullah, Arch. Biochem. Biophys., Vol. 137, 483 (1970) etc.], Streptomyces [S. Ueda et. al., J. Ferment. Tech., Vol. 49, 552 (1971) etc.], Streptococcus [Streptococcus mitis, G. W. Walker, Biochem. J., Vol. 108, 33 (1968) etc.], Escherichia [Escherichia intermedia, S. Ueda et. al. Applied Microbiol., Vol. 15, 492 (1967), U.S. Pat. No. 3,716,455(1973) etc.], Bacillus [Japanese Patent Application Kokoku 25036/1987, 25037/1987, Agric. Biol. Chem. Vol. 40, No.8, 1523 (1976) K. Horikoshi Japanese Patent Application Kokoku 27786/1978 etc.], Flavobacterium [Flavobacterium esteromaticum, Japanese Patent Application Kokoku 18826/1973], Cytophaga [U.S. Pat. No. 3,790,446 (1974), Lactobasillus, Micrococcus, Nocardia, Staphylococcus, Azotobactger, Sarcina etc. [England Patent 11260418, U.S. Pat. No. 3,827,940 (1974)] and Actinomycetes [Norcardia, Micromonospora, Thermomonospora, U.S. Pat. No. 3,741,873 (1973)].
Most of the pullulanase known in the art are not heat stable and its optimum temperature is 40.degree.-50.degree. C. Even if pullulanase is heat stable, it has problem in pH: the working pH of a pullulanase is limited in an acidic side (optimum pH: 5) and the pullulanase does not work at neutral pH (e.g., pullulanase produced by bacteria belonging to the genus Bacillus [Japanese patent application kokoku Sho 62-25036, 62-25037]; the working pH of a pullulanase is limited at around neutral pH and the pullulanase does not work at acidic side (e.g., pullulanase produced by bacteria belonging to the genus Streptomyces [J. Ferment. Tech., Vol. 49, 552 (1971) etc.]). These pullulanase for one reason or another have characteristics which are not favorable as an all-purpose pullulanase for industrial use.